Large-Scale Analysis of Fitness Cost of tet(X4)-Positive Plasmids in Escherichia coli

Tigecycline is one of important antimicrobial agents for the treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, the emergence and prevalence of plasmid-mediated tigecycline resistance gene tet(X4) are threatening human and animal health. Fitness cost elicited by resistance plasmids is a key factor affecting the maintenance and transmission of antibiotic resistance genes (ARGs) in the host. A comparative analysis of the fitness cost of different types of tet(X4)-positive plasmids is helpful to understand and predict the prevalence of dominant plasmids. In this study, we performed a large-scale analysis of fitness cost of tet(X4)-positive plasmids origin from clinical isolates. These plasmids were successfully electroporated into a reference strain Escherichia coli TOP10, and a series of transformants carrying the tet(X) gene were obtained. The effects of tet(X4)-positive plasmids on the growth rate, plasmid stability, relative fitness, biofilm formation, and virulence in a Galleria mellonella model were evaluated. Consequently, we found that these plasmids resulted in varying degrees of fitness cost on TOP10, including delayed bacterial growth and attenuated virulence. Out of these plasmids, tet(X4)-harboring IncFII plasmids showed the lowest fitness cost on the host. Furthermore, by means of experimental evolution in the presence of commonly used drugs in clinic, the fitness cost of tet(X4)-positive plasmids was substantially alleviated, accompanied by increased plasmid stability. Collectively, our data reveal the differential fitness cost caused by different types of tet(X4)-positive plasmids and suggest that the wide use of tetracycline antibiotics may promote the evolution of plasmids.